Cytotoxic Activity of Saponins and Sapogenins Isolated from Chenopodium quinoa Willd. in Cancer Cell Lines

The cytotoxic properties of two extracts from Chenopodium quinoa Willd. and three synthetic sapogenins were evaluated in different cancer cell lines (A549, SH-SY5Y, HepG2, and HeLa) to investigate their cytotoxic effects and determine if these cell lines activate the caspase pathway for apoptosis in response to saponin and sapogenin treatment. The saponin extracts were isolated from the agro-industrial waste of Chenopodium quinoa Willd., while the sapogenins were identified and quantitatively determined by High-Performance Liquid Chromatography (HPLC). Among these compounds, ursolic acid was the most active compound, with high IC50 values measured in all cell lines. In addition, hederagenin demonstrated higher caspase-3 activity than staurosporine in HeLa cells, suggesting an anti-cytotoxic activity via a caspase-dependent apoptosis pathway. HPLC analysis showed that the concentration of hederagenin was higher than that of oleanolic acid in ethanolic extracts of white and red quinoa. The ethanolic extracts of white and red quinoa did not show cytotoxic activity. On the other hand, the synthetic sapogenins such as ursolic acid, oleanolic acid, and hederagenin significantly decreased the viability of the four cell lines studied. Finally, by Caspase-3 assay, it was found that HeLa undergoes apoptosis during cell death because hederagenin produces a significant increase in PARP-1 hydrolysis in HeLa cells.


Introduction
Te World Health Organization has reported that cancer is one of the ten most frequent causes of death worldwide, with a drastic increase in its incidence.Given that conventional modalities for the treatment of cancer are invasive and expensive, new alternatives have been proposed and the interest in medicinal plants as possible sources of compounds with anticancer activity has increased.Quinoa grain (Chenopodium quinoa Willd.) is known for its high nutritional content which is a functional and ideal food for human beings.Quinoa grain contains 18% protein and is a rich source of fber, antioxidants, and minerals; moreover, it is gluten-free and contains vitamins such as A, B2, C, E, thiamine, and folic acid.Te fats present in quinoa include omega 6, omega 9, omega 3, and palmitic acid, and it contains between 58 and 68% carbohydrates [1][2][3][4][5].However, quinoa contains several compounds that decrease its nutritional quality, one of which is the saponin located in the pericarp of the seeds.Saponins are abundant antinutrients that are intensely bitter compounds and potentially toxic if ingested in large quantities and thus function as protection against birds, insects, and fungi [6].
Te varied composition of Chenopodium quinoa Willd.could provide this food with diferent properties such as an anticarcinogenic efect, since it has been demonstrated that the phenolic compounds of quinoa have an inhibitory efect on the proliferation of AT-2 and MAT-LyLu rat prostate cancer cells [7].Another study demonstrated that quinoa proteins exhibit viability inhibitory properties in human colorectal cancer cell lines (Caco-2, HT-29, and HCT-116) [8].Likewise, quinoa seed powder showed cytotoxicity against the hepatocarcinoma cell line HEPG2 and would provide hepatoprotection against non-alcoholic fatty liver disease [9].Also, quinoa protein or its hydrolysate was shown to improve the azoxymethane/sodium dextran sulfate-induced colorectal cancer mouse model by altering the intestinal fora and increasing the production of benefcial short-chain fatty acids [10].On the other hand, a new polysaccharide made up of galacturonic acid and glucose isolated from quinoa showed an anticancer efect in human liver cancer SMMC 7721 and breast cancer MCF-7 cells [11].Black quinoa seed oil had an antiproliferative efect on HCT 116 (human colon carcinoma) cells by inducing apoptosis in a dose-dependent manner [12].Finally, the aglycones of quinoa saponins that are triterpenoids called triterpenoid saponins would show anticancer and anti-infammatory efects [13,14].
Quinoa contains at least 30 diferent types of saponins derived from sapogenins such as hederagenin, oleanolic acid, serjanic acid, and phytolaccagenic acid [6,15,16].Because these compounds possess broad properties, the objective of this research was to evaluate the cytotoxic efects of saponins and sapogenins isolated from Chenopodium quinoa Willd.on diferent cancer cell lines to provide a basis and additional information for future preclinical cancer research.

General Experimental Procedures.
All solvents and reagents were purchased from commercial sources and used directly without further purifcation.Te starting plant material was provided as a fne powder of two types of Chenopodium quinoa Willd.saponins (red and white quinoa), obtained by the company JIWRA SAC (Arequipa, Peru).High-Performance Liquid Chromatography (HPLC) was conducted on a Hitachi Primaide with a DAD detector Primaide 1430 and a Termo Scientifc ™ Hypersil GOLD ™ C18 Reversed Phase HPLC Column, 5 μm, 2.1 mm × 20 mm.Te purity of all compounds was >95% as determined by HPLC.Methanol : acid water (89 : 11) was used as a mobile phase with a fow rate of 1.5 mL/min.Te Tin Layer Chromatography (TLC) analysis was carried out on silica gel plates that were visualized under UV at 366 nm and sprayed with Liebermann-Burchard reagent before heating.

Preparation of Plant Extracts.
Extraction of the active compounds of Chenopodium quinoa Willd.was performed using a percolation method with 95% ethanol.Both plant samples were treated separately.Te plant matrix was suspended in the solvent for 24 h, and then the extracts were drained at a fow rate of 60 drops/min.Finally, the ethanol was evaporated with a rotary evaporator system (B ÜCHI).

Caspase-3 Activity.
Te caspase-3 activity in A549, HeLa, SH-SY5Y, and HepG2 cells was measured with the caspase-3 colorimetric assay kit (BioVision K-106) [22].After treatment with the staurosporine [23] positive control and the three synthetic sapogenins at diferent concentrations, the four cell lines were harvested and lysed on ice.Te protein in the cell lysates was quantifed using the BCA (bicinchoninic acid) assay and adjusted to the suggested concentration before the caspase activity was measured.Caspase-3 recognizes the DEVD sequence and cleaves the provided DEVD-pNA substrate, which produces the pnitroaniline chromophore that emits a quantifable light signal that can be measured at 405 nm by a spectrophotometer.All assays were performed in triplicate.

Western Blotting Analysis.
Half a million HeLa cells were seeded in 6-well culture dishes and were treated with different concentrations of ursolic acid (UA), oleanolic acid 2 Scientifca (OA), or hederagenin (HED) for 5 and 24 h.Te medium was then removed and the cells were washed twice with 500 μL of cold PBS (phosphate bufered saline) (4 °C) before 300 μL of MPER cell lysis reagent (Termo Fisher Scientifc) was added to each well and incubated for 5 min at 4 °C.Te wells were scraped to collect the cells, which were transferred to microcentrifuge tubes.Lysed proteins were isolated by centrifugation and quantifed using a BCA assay.Te same volume of proteins was separated by electrophoresis on a 7.5% polyacrylamide gel (Bio-Rad) and then subjected to electroblotting on a 0.45 µm pore diameter nitrocellulose membrane.Te blots were probed with anti-cleaved PARP-1 (poly(ADP-ribose) polymerase-1) (Abcam; 1 : 5000), anti-PARP-1 (Abcam; 1 : 5000), β-actin (Abcam; 1 : 10000), and goat polyclonal antibody to rabbit IgG (HRP) (Abcam; 1 : 5000).Reactive bands were detected by chemiluminescence on a C-DiGit blot scanner (LI-COR Biosciences).Images were captured, stored, and analyzed with the "Image Studio Digits ver.5.0" program.

Obtaining Saponins via Liquid-Liquid Extraction.
Five grams of the plant sample concentrate were weighed and dissolved in 15 mL of distilled water and poured into a decantation funnel where it was subjected to three continuous extractions with 10 mL of chloroform each.Te aqueous phase was recovered and extracted three times with 10 mL of ethyl acetate each to eliminate the oily phase.Finally, three extractions with 1-butanol were done to eliminate the aqueous phase.Te oily phase was collected and placed in an oven at 24-90 °C until complete dryness.Te powder obtained was then dissolved in 10 mL of 1% NaOH, and three more extractions were conducted with 5 mL of 1-butanol.Te butanol fractions were put back into the oven set to the conditions indicated above to obtain the purifed saponins.Both isolates from white and red quinoa were subjected to this procedure.

Percolation of Sapogenins by Acid Hydrolysis.
One hundred milligrams of saponins isolated from red and white quinoa were weighed separately and dissolved in 25 mL of distilled water and heated in a water bath; 10 mL of concentrated hydrochloric acid was added to refux for 20 min.
Te pH reaction was adjusted with 30% sodium hydroxide before the solutions were added to a decantation funnel in which three successive extractions were carried out with 10 mL of chloroform.Te chloroform phase was collected and placed in an oven until the solvent was completely evaporated and a dry powder was obtained.
2.9.HPLC Analysis.Hederagenin and oleanolic acid stock solutions of 2, 4, 8, 16, and 32 mg/L were used to calibrate the system.Additionally, 600 µL of saponins isolated from each of the quinoas was added to 2 mL of methanol and was fltered through a glass syringe into sample vials.
Troughout the development of the experiment, ≥99% HPLC-grade methanol was used.Methanol : acid water (89 : 11) was used as a phase solution for HPLC experiments.It was prepared by diluting orthophosphoric acid in ultrapure water, to which 20 µL of each synthetic sapogenin was loaded into a GOLD Hypersil-5 µm column C-18 (Termo Fisher Scientifc), with a 10 min run at 20 °C and 1.5 mL/min fow rate.Each run was performed in triplicate, and the wavelength for the detection of hederagenin and oleanolic acid was 210 nm.

3.1.
In Vitro Cytotoxic Activity of Chenopodium quinoa Willd.Ethanolic Extracts.Te cytotoxic activity of the ethanolic extracts from red and white quinoa was evaluated in four diferent cancer cell lines using a concentration range of 0-100 mg/L in an MTS assay.Te results demonstrated that neither of the two ethanolic extracts reduced cell viability in the cell lines tested (data not shown), indicating that both the white and red quinoa extracts had no cytotoxic efect, and therefore we did not test further.

Te Efect of Ursolic Acid on the Cell Viability of A549,
HeLa, SH-SY5Y, and HepG2 Cell Lines.Te efect of ursolic acid (UA) on cell viability was evaluated using the MTS assay in four cancer cell lines.Te results are presented as the concentration of UA that inhibited the growth of the cells by 50% (IC 50 ).Te cell viability was reduced in cancer cell lines treated with 0-300 µM UA, suggesting that UA had a cytotoxic efect at concentrations of 30 µM, 10 µM, 300 µM, and 10 µM on A549, HeLa, HepG2, and SH-SY5Y cell lines, respectively (Figure 1).Te IC 50 values were 21.9 ± 0.05 μM for A549, 11.2 ± 0.05 μM for HeLa, and 104.2 ± 0.05 μM for HepG2, with the highest activity of 6.9 ± 0.05 µM in SH-SY5Y cells.

Efect of Oleanolic Acid on Cell Viability in Cancer Cell
Lines.Te efect of OA on the cell viability of four diferent cancer cell lines was evaluated using the MTS assay using concentrations of 0 µM to 300 µM OA.Tis sapogenin displayed cytotoxic activity in the A549, HeLa, HepG2, and SH-SY5Y cell lines at concentrations equal to or greater than 100 µM (Figure 2).Tese results indicated that this efect of the mentioned compound increased in a dose-dependent manner.Moreover, the lowest cytotoxic activity was found in HepG2, A549, and HeLa cells, with IC 50 of 408.3 ± 0.05 µM, 98.9 ± 0.05 µM, and 83.6 ± 0.05 µM, respectively.Te highest activity was observed in SH-SY5Y cells, with IC 50 of 34.1 ± 0.05 µM.

Efect of Ursolic Acid, Oleanolic Acid, and Hederagenin, on
Caspase-3 Activity Cancer Cell Lines.Te results from the treatment of cancer cell lines with UA, OA, and HED sapogenins encouraged us to evaluate whether A549, HeLa, HepG2, and SH-SY5Y cell lines activate the caspase pathway for apoptosis in response to saponin and sapogenin treatment.To verify that the four cell lines do activate the caspase pathway for apoptosis, a preliminary test was performed.Each of the lines was treated with 1 µM STS and found to have higher caspase activity in the positive control (STS) when compared with that in the untreated cells (Figure 4(a)).Te only cell line in which no activity was observed even when treated with high concentrations of STS (100 µM) was A549; therefore, it was excluded from subsequent treatments.
On the other hand, the SH-SY5Y, HeLa, and HepG2 cell lines that did present caspase-3 activity were treated at determined concentrations of OA, UA, and HED which were established according to the IC 50 results obtained from the cell viability assay presented in Table 1.We found that the only cell line that had activity compared to the positive control was HeLa, with concentrations of 100 µM of OA, 30 µM of UA, and 100 µM of HED, respectively, and therefore, SH-SY5Y and HepG2 were not used for further experimentation (Figure 4(a)).
To evaluate the caspase-3 activity in HeLa cells, additional concentrations of the three sapogenins were used as treatments.A one-way ANOVA test and an unpaired Student's t-test were used to evaluate the efects of these treatments on caspase-3 activity in these cells.We found that there was a signifcant increase in caspase-3 production when the cells were treated with the positive control (STS) compared to untreated control cells (Figure 4(b)), thus confrming that this potent, nonselective inhibitor of protein kinases induces apoptosis as a mechanism of cell death [24].Furthermore, although the treatment with OA induced signifcant dose-dependent caspase activity (p < 0.003), the efect was mild.While only treatment with 100 µM UA showed signifcant activity when compared to the control, HED had the most signifcant efect on caspase-3 activity in the HeLa cell line (p < 0.02).4 Scientifca

Sapogenins Participate in the Activation of Apoptotic
Proteins in HeLa Cells.Western blotting analysis of HeLa cells treated with UA, OA, and HED for 5 h (Figure 5(a)) showed a series of faint bands for PARP-1 (113 kDa), as well as those for the β-actin loading control (42 kDa).Cleaved PARP-1 (25 kDa) bands had greater intensity in samples from cells treated with 100 µM UA and OA when compared with the positive control (cells treated with STS).Te other treatments that were evaluated included 300 µM OA, 50 µM, 100 µM, and 300 µM of the HED, apoptosis-inducing positive control (STS), and a negative control (untreated cells).Both UA and OA treatments induced twice as much apoptosis compared to the control at a concentration of 100 µM, indicated by the increased expression of cleaved PARP-1 (Figure 6(a)).Tese results suggested that caspase-3 cleaved PARP-1 in the late phase of apoptosis.Additionally, when HeLa cells were treated with 100 µM HED, apoptotic activity was three times higher compared to that in the positive control, which is the highest activity observed of the conditions tested and indicated that among all the sapogenins studied, HED had the greatest capacity to induce cell death via apoptosis.
Figure 5(b) presents the results of Western blot analysis of HeLa cells treated with 100 μM UA, 100 μM, and 300 μM OA and 50 μM HED for 24 h.Some of the concentrations after 5 h were excluded because the treatments after 24 h had killed all the cells and the necessary amount of protein could not be obtained for evaluation.Concentrations of 300 µM OA and 50 µM HED did not induce apoptosis after 24 h, indicated by the lower expression of cleaved PARP compared with the control (Figure 6(b)).Conversely, caspase-3 cleaved PARP-1 indicated higher apoptotic activity in cells treated with 100 µM UA and OA than the negative and positive controls.

Tin Layer Chromatography Profle of Saponins and
Sapogenins from Red and White Quinoa Extracts.To demonstrate the presence of saponins in quinoa extracts, we used TLC to separate the biochemical contents of both samples of white and red quinoa extracts.Figure 7 shows the TLC plates on which various spots indicated that the samples were not purifed; however, a pink-purple spot was present in the

Scientifca
white quinoa (QB) sample, while an intense violet-colored spot was in the red quinoa (QR) sample.Tese results are consistent with triterpenoid saponin [25].Te plate was exposed to 366 nm light, which did not show the saponin spots; however, a Retardation Factor (Rf ) of 0.46 was obtained for QB and that of 0.58 was obtained for QR.
We also performed TLC to identify the sapogenins after acid hydrolysis using AU, HED, and AO sapogenins (Figure 7(c)).Ursolic acid was observed as a light violet color with a Rf = 0.66 while HED has a violet color with a Rf of 0.38, and OA had a Rf equal to that of UA.Te quinoa samples coincided with UA and OA, as well as HED.Te Rf values of HED and OA in red quinoa were 0.38 and 0.66, respectively, which were similar to those of white quinoa (Rf = 0.38 for HED and Rf = 0.67 for OA).

Determination of AO and HED in Chenopodium quinoa
Willd.Extracts.We conducted HPLC runs in triplicate for each sapogenin and obtained the calibration curve (Figure 8) that enabled us to fnd the concentrations of sapogenins either AO or HED, for each gram of powder in both kinds of quinoa.OA and HED were 5.61 ± 0.02 mg/L (0.0123 µM) and 9.43 ± 0.02 mg/L (0.020 µM), respectively, in white quinoa.In red quinoa, the HPLC results showed that there were 5.30 ± 0.01 mg/L (0.0116 µM) of OA and 14.44 ± 0.01 mg/L (0.0305 µM) of HED.Tese results indicated that for every gram of powder residue that is produced when processing white quinoa, there are 2.805 mg of OA and 4.715 mg of HED.Likewise, for each gram of powdery residue that is produced when processing red quinoa, there are 2.65 mg of OA and 7.22 mg of HED.

Discussion
In this report, we evaluated the cytotoxic activity of ethanolic extracts obtained from white and red quinoa in A549, SH-SY5Y, HepG2, and HeLa cell lines.Our fndings demonstrated that both white and red quinoa extracts did not afect the viability of these cells.However, the synthetic sapogenins showed that UA reduced the cell viability percentage at  6 Scientifca lower concentrations when compared to OA and HED.Furthermore, the IC 50 values for cells treated with UA were lower compared to OA and HED (Table 1).It has been reported that in HCT15 cells treated with the same concentrations of UA and OA, UA could reduce cell viability more than OA.Similarly, other investigations confrmed the cytotoxic capacity of UA and its derivatives in HL-60, BGC-823, Hela, and Bel-7402 cells [26][27][28].While evaluating caspase-3 activity in cell lines treated with saponins, we observed that A549 cells did not activate caspase-3, consistent with previous investigations that reported that this cell line uses autophagy to mediate cell death [29,30].Furthermore, we observed that HeLa cells had the highest caspase-3 activity compared to the control (Figure 4(a)).Likewise, analysis in HeLa cells indicated that HED could markedly increase the activity of caspase-3.Previous studies have reported that triterpenoid sapogenins extracted from plants can be cytotoxic in HeLa cells in a caspase-dependent manner [31][32][33].Additionally, natural substances isolated from plants have demonstrated cytotoxic activity against cancer cell lines, as demonstrated in a study where they found that the natural favonoid fsetin induced apoptosis of HeLa cells by triggering the activation of caspases-3 and -8 and the cleavage-PARP, resulting in the induction of apoptosis [34,35].
When evaluating cleaved PARP-1, HED promoted the highest cleaved PARP-1 activity of the saponins tested.PARP-1 is responsible for DNA damage repair and it is one of the target substrates of caspase-3 [22].Terefore, the 8 Scientifca higher the percentage of cleaved PARP-1, the greater the apoptotic activity.Additionally, it was reported that this saponin increased the levels of pro-apoptotic proteins such as cleaved PARP and BAX in head and neck cancer cell lines [36].By knowing the concentration in mg/L of the sapogenins in both quinoa extract samples, a conversion was made from the concentration units used in the HPLC (mg/L) to the units used in the cytotoxicity assays (µM) to compare the concentrations of sapogenins in extracts versus those that caused a cytotoxic efect in the cell lines studied.Te results obtained to explain the lack of cytotoxicity activity of the ethanolic extracts showed that the concentrations of sapogenins were less than 1 µM, while minimum concentration of 12.30 ± 0.05 µM (Table 1) was necessary to afect viability in these cell lines.

Conclusions
Te ethanolic extracts were obtained from the residues of Chenopodium quinoa Willd.and they did not present cytotoxic activity.However, the synthetic sapogenins: UA, OA, and HED, signifcantly decreased the viability of HeLa, A549, HepG2, and SH-SY5Y cancer cell lines.Similarly, through the caspase-3 assay, it was determined that HeLa undergoes apoptosis during cell death.However, the mechanisms of cell death in lines A549, HepG2, and SH-SY5Y are unknown, and further studies are needed.It was determined that ethanolic extracts of red and white quinoa had higher concentrations of HED than OA.On the other hand, it was found that HED produced a signifcant increase in PARP-1 hydrolysis in HeLa cells.It would be important to examine in greater depth the function of hederagenin in each of the cell lines, fow cytometry could be implemented to obtain a better cell count during viability, and the use of cellular biomarkers would help to have better information on the biological state of the cell.Taking into account the present research, future studies could focus on studying the composition of the husks of diferent quinoa varieties since they are wastes of this food that could be a source of sapogenins with potential cytotoxic activity in cancer cells.

Table 1 :
IC 50 values of cancer cell lines after saponin and sapogenin treatment.

Figure 8 :
Figure 8: Hederagenin (blue line) and oleanolic acid (red line) calibration curve.Te diferent concentrations were compared against the specifc area shown during the HPLC run each sapogenin.